Sucrose phosphate synthetase (SPS), its preparation process, its complementary DNA and the use of the complementary DNA to modify the expression of the SPS in vegetable cells.
The present invention relates to saccharose phosphate synthetase (SPS), its preparation process, its complementary DNA and the use of the latter to modify the rate of expression of SPS in vegetable cells.
A subject of the invention is proteins having the activity of saccharose phosphate synthetase (SPS).
By vegetable cell is meant all plant cells being able to form undifferentiated tissue such as calluses or differentiated tissues such as embryos, certain parts of plants, whole plants or also seeds.
By plant is meant notably plants producing seeds, for example the grasses such as straw cereals such as wheat, barley, maize or oats, the legumes such as soya, oleaginous plants such as sunflowers, plants with tubers such as potatoes, plants with roots such as beetroots or fruits such as tomatoes.
More particularly a subject of the invention is saccharose phosphate synthetase and notably the saccharose phosphate synthetase of plants.
By plants is meant for example the grasses such as for example wheat, barley, maize, sugar cane, vegetables such as tomatoes and soya, fruits, such as apples and bananas.
Saccharose phosphate synthetase is a key enzyme in the regulation mechanisms of saccharose, but also in the regulation mechanisms of the distribution of carbon between starch and saccharose in photosynthesis (see on this subject the article by Jack PREISS in TIBS January 1984, pages 24 and following, or also the article by Mark STITT and Coll. in Biochemistry of Plants vol. 10, 1987 pages 327 and following).
The SPS appears to be specific to the species concerned; Joan L-Walker and Steven C. Huber who have purified and carried out a preliminary characterization of the saccharose phosphate of spinach indicate clearly that the antibodies obtained recognize exclusively the SPS of spinach (cf PLANT PHYSIOL (1989) 89, 518-524).
More precisely a subject of the invention is the SPS of maize.
The SPS of maize can exist in a pure or practically pure form.
More precisely a subject of the invention is the proteins defined previously of molecular weight of the order of 110 to 130 kD appearing in the monomer, dimer or tetramer form and their derivatives having at least one peptide whose amino acid sequence is the following:
ThrTrpIleLys (SEQ ID NO: 1)
TyrValValGluLeuAlaArg (SEQ ID NO: 2)
SerMetProProIleTrpAlaGluValMetArg (SEQ ID NO: 3)
LeuArgProAspGlnAspTyrLeuMetHisIleSerHisArg (SEQ ID NO: 4)
TrpSerHisAspGlyAlaArg (SEQ ID NO: 5)
Notably a subject of the invention is the proteins defined previously having the amino acid sequence described in FIG. 7. (SEQ ID NO: 11)
Also a subject of the invention is the derivatives of the proteins defined previously modified by genetic engineering techniques and presenting the activity of the SPS.
Also a subject of the invention is a preparation process characterized in that:
a) an extract is made from parts of the plants preserved at low temperature by grinding, centrifugation and filtration,
b) the extract obtained is enriched in SPS protein by precipitation in an appropriate solvent, centrifugation and solubilisation of the precipitate obtained in a buffer solution,
c) the active protein thus obtained is purified by chromatography and if desired,
d) the hybridomas and monoclonal antibodies are prepared from an antigen solution obtained from one of the preparations obtained in paragraphs a), b), and c) above,
e) the hybridomas are screened and the monoclonal antibody or antibodies directed specifically against the SPS are selected,
f) the SPS obtained is purified by means of the antibodies prepared in this way.
More precisely a subject of the invention is a process characterized in that:
a) an extract is made from parts of maize plants preserved at low temperature by grinding, centrifugation and filtration,
b) the extract obtained is enriched in proteins by precipitation in polyethyleneglycol, centrifugation and solubilisation of the precipitate obtained in a buffer solution,
c) the SPS protein thus obtained is purified by low pressure anion exchange chromatography, then by chromatography on heparin Sepharose, then by high pressure anion exchange chromatography,
d) the active fractions are purified by passage through 2 high pressure chromatography columns, and, if desired,
e) the hybridomas and monoclonal antibodies are prepared from an antigen solution obtained from a preparation a), b), c),
f) the hybridomas are screened and the antibodies directed specifically against the SPS are selected,
g) the SPS obtained previously is purified by means of antibodies thus prepared.
In a preferred embodiment:
the maize used is a maize of PIONEER 3184 strain,
the parts of maize plants used are leaves preserved at low temperature for example between xe2x88x9250xc2x0 C. and xe2x88x9290xc2x0 C.,
the purification in polyethyleneglycol (PEG) takes place in two stages:
a first precipitation where the final concentration of PEG is close to 6%,
a second precipitation where the final concentration of PEG is close to 12%.
the various chromatographies are carried out as follows:
1st chromatography: DEAE Sepharose,
2nd chromatography: heparin Sepharose: it should be noted that the preparation thus obtained can be preserved for several days without a major loss of activity,
3rd HPLC chromatography: Mono Q chromatography,
4th HPLC chromatography: hydroxylapatite,
5th HPLC chromatography: DEAE.
in the course of these various purification stages and the following, the measurement of the SPS activity is preferably carried out using two different methods:
a) a method based on a colorimetric test or a resorcinal test,
b) a method based on the determination of one of the products formed during the transformation reactions involving the SPS. These two methods are detailed in the experimental part set out hereafter.
mice are immunized with several injections of the purified enzymatic preparation.
Different mouse types can be used, for example BALB/C mice.
The antigen is used in the complete Freund adjuvant then in the incomplete Freund adjuvant.
Several injections of the antigen are administered to the mice: good results were obtained with three injections of mono Q fractions followed by three injections of final fractions (on days 0, 14, 27, 60, 90 and 105 for example).
The first injections are carried out by subcutaneous route, for example in the foot pads, the last injection is carried out by intravenous route in the tail for example.
the preparation of cellular suspensions of spleen thus immunized is treated in a clonic fashion.
The stages of fusion with myeloma cells, preservation of the hybridomas, cloning and production of the antibodies are achieved according to known methods.
To detect the hybridomas secreting the antibodies directed against the antigen, two methods are used to select the detection antibodies of the secreting hybridomas directed against the immunization antigen:
a method of detection of antibody inhibitors of SPS activity,
a method of antibody activity directed against SPS activity.
These methods are preferably those described in the experimental part.
Also a subject of the invention is the cell lines of the hybridomas obtained and notably the cell lines of the following hybridomas:
SPA 2-2-3: I-971
SPA 2-2-22: I-970
SPA 2-2-25: I-972
SPB 3-2-19: I-973
SPB 5-2-10: I-974
SPB 5-4-2: I-975
SPB 13-1-7: 1-976
SPB 13-2-2: I-977
which were deposited on Jun. 11, 1990 with the Collection Nationale de Culture de Microorganismes (CNCMxe2x80x94Institute Pasteur) under the numbers mentioned located at 28, rue de Docteur Roux, 75724 Paris Cedex 15, France.
Also a subject of the invention is monoclonal antibodies directed specifically against the SPS.
Also a subject of the invention is a preparation process for proteins characterized in that a preparation containing said proteins is passed through a chromatography column containing the monoclonal antibodies directed specifically against said proteins and in this way the desired proteins are obtained. Also a subject of the invention is the DNA coding sequences for the proteins defined previously and notably the SPS of maize, whose sequence appears in FIG. 7.
The complementary DNA (cDNA) coding for the saccharose Phosphate Synthethase (SPS) enzyme was prepared as follows:
1xe2x80x94Sequencing of purified SPS peptide fragments.
Purified preparations of maize SPS obtained previously give, upon separation on acrylamide gel, a minor band of 120 kd (corresponding to the total protein sequence) and two major bands of 90 and 30 kd. These two polypeptides are separated by electrophoresis then electroelution. Trypsic digestion followed by the sequencing of fragments obtained allowed the amino acid sequences of 5 peptides to be determined (FIG. 3). Knowledge of the amino acid sequence of these peptides allows the corresponding nucleotide sequence to be determined.
2xe2x80x94Isolation of the RNA of maize leaves.
The total RNA is isolated according to the TURPEN and GRIFFITH method (1986, Biotechniques vol.4 pp 11-15).
The RNA polyA+ is prepared by passage through a column of oligodT cellulose according to known techniques.
3xe2x80x94Construction of a cDNA library.
The cDNA synthesis is carried out using the xe2x80x9cPROMEGAxe2x80x9d(copyright) synthesis kit. The reverse transcriptase of MMLV is used in place of AMV reverse transcriptase. The size of cDNA""s obtained is comprised between 500 base pairs and several thousand base pairs. ECoRI adapters are added to the ends of the cDNA before cloning in a lambda gt11 expression vector. The cDNA library contains approximately 1.5xc3x97106 transformants.
4xe2x80x94The use of PCR for the synthesis of a specific nucleotide probe for SPS.
The oligonucleotides derivatised from B11 peptide sequences (coming from 30 kd) and 4 K (coming from 90 kd) described in FIG. 3 are used as initiators in PCR-type reactions. The starting hypothesis is that the polypeptides of 30 and 90 kd are the degradation products of the SPS protein of 120 kd. Therefore the peptides resulting from the SPS 30 and SPS 90 fragments have to come from the translation of the same RNA messenger. In this hypothesis the use of a pair of oligonucleotides corresponding to the peptide sequences in a PCR-type reaction has to result in the synthesis of a DNA fragment of determined size if these oligonucleotides are complementary to an indentical DNA sequence. Not knowing the respective position of those peptides in the SPS protein, the various combination are tried out. Only the pair of CD3 oligonucleotides (FIG. 4) gives a DNA fragment of determined size (1200 base pairs).
5xe2x80x94Screening the cDNA library.
250,000 lambda gt11 transformants have been screened using the DNA fragment of 1200 base pairs obtained by PCR reaction (described previously). 16 positive clones have been obtained. The insertion sizes vary from 0.3 kb to 2.8 kb. The Sequence obtained is not complete on the Rxe2x80x2 side. A second screening of the library using a DNA fragment of 400 bp corresponding to the 5xe2x80x2 part of the SPS3 clone allows a clone (SPS 61) to be obtained which goes further in the 5xe2x80x2 part (FIG. 6) without having the 5xe2x80x2 terminus.
6xe2x80x94Production and screening of a second cDNA library allowing the cloning of the 5xe2x80x2 part of the cDNA coding for the SPS.
An oligonucleotide complementary to the 5xe2x80x2 sequence of the SPS61 clone is used as initiator for the synthesis of the cDNA. After synthesis of the second strand, the cDNA was cloned in the lambda phage. The library contains approximately 1 million clones. The SPS 90 and SPS 77 clones were obtained during the screening of this library with SPS 61 (FIG. 6). The sequence of these clones has allowed the region overlapping with the SPS 61 clone to be determined. The SPS 90 clone allows the 5xe2x80x2 part of the SPS to be reached.
Verification of the organization of the different sequences (FIG. 6) allowing the complete cDNA sequence to be obtained was able to be carried out by using the PCR technique. The initiators used belong to the SPS 3 and SPS 90 clones. The obtaining of a fragment of 750 base pairs of the exact size predicted by the complete sequence permits the assertion that the SPS 3 and SPS 90 clones derive from the same RNA messenger.
7xe2x80x94Assembly of the complete cDNA.
Also a subject of the invention is the genomic DNA containing the coding part for the proteins defined previously and sequences necessary for the expression and regulation of this protein in plants.
Also a subject of the invention is a process for modifying the expression rate of the SPS in a plant, characterized in that the cells of said plant are transformed by means of an expression vector containing the cDNA defined previously.
Also a subject of the invention is a vector allowing the expression of the SPS protein under the control of a promoter capable of directing the expression and preferably the superexpression of said SPS in a plant cell and a 3xe2x80x2 region containing transcription regulation signals for the expression of the gene coding for the SPS.
Moreover a subject of the invention is the plants obtained by the implementation of this process.
Also a subject of the invention is the seeds obtained.